Hemoglobin cross-linked by bis (3,5-dibromosalicyl) fumarate has a higher oxygen-carrying capacity as well as longer half-life than extracted intact hemoglobin. This cross-linked material is therefore a valuable blood substitute. Baxter Biotech has been developing this product for a long period and has now reached a stage that a detailed characterization of the product is required. This task will be carried out using mass spectrometric based techniques, taking advantages of the speed and sensitivity of mass spectrometry. The joint project between Baxter and UCSF will use this technique as the primary means to solve this problem. Experiments will include 1. LC/ESIMS to recognize all kinds of modifications; 2. Enzymatic digestion and HPLC separation of peptides; 3. MALDI/PSD for molecular weight and sequence; and 4. ESI/MS/MS or MALDI/MS/MS for sequence information of modified peptides. These experiments will provide necessary information for understanding the nature of the major and minor products of the cross-linking sites of the major and minor fractions. Cross-linking between hemoglobin molecules will also be analyzed.